The term “cancer” comprises a heterogeneous group of neoplasms, in which each type has its own characteristics when considering its malignant potential and its response to therapy. It goes without saying that accurate diagnosis and classification of the various cancer types is pre-eminent in helping to select the most effective therapy. Furthermore, a diagnostic method allowing the detection of small numbers of malignant cells in a high background of normal cells during therapy is essential for evaluating treatment effectiveness and for anticipating an impending relapse.
Chromosomal translocations can be detected by a wide array of techniques, most of which entail modern biomolecular technology. Cytogenetic techniques include conventional chromosomal banding techniques (karyotyping) and fluorescence in situ hybridization (FISH) which uses fluorescently labeled probes. Polymerase chain reaction-(PCR-) based strategies can be used to detect fusions of chromosomal breakpoints as can be found in chromosomal translocations, inversions and deletions using primers located at each side of the breakpoint. DNA amplification can only be used for chromosome aberrations in which breakpoints cluster in a small area. In most cases, breakpoints spread over large intronic regions, but several translocations, inversions and deletions give rise to fusion genes and fusion transcripts suitable for PCR amplification after a reverse transcription step (RT-PCR).
Most commonly used techniques aimed at detecting specific chromosomal aberrations involve analysis at the chromosomal or nucleic acid (DNA or RNA) level. An advantage of such genetic fusion markers is their direct involvement in oncogenesis. Accordingly, their presence is constant all over disease evolution. However, a major drawback of fusion markers relates to the fact that variations in the level of gene transcription and/or gene translation during the disease and particularly during therapy cannot be excluded. Thus, variations in expression of a fusion gene transcript or a fusion protein make it difficult to correlate the level of detection of the marker to the amount of malignant cells. This implies that detection of a fusion gene product is preferably performed at the protein level in individual cells.
A fusion protein comprises parts of at least two proteins that correspond to, and were originally transcribed by and translated from, the originally separated genes. Fusion proteins are uniquely characterized by a fusion point where the two proteins meet. Fusion points are often antigenically exposed, comprising distinct epitopes that sometimes can be immunologically detected.
Initially, attempts were made to raise fusion protein-specific antibodies by generating antibodies against a peptide corresponding to the joining region of a fusion protein. This approach has rarely been successful, mainly because of the fact that it is difficult to find immunological reagents that are exclusively reactive with a fusion protein and not with the non-fusion proteins that are normally produced in a cell. If fusion-specific antibodies were obtained, they were generally not applicable to fluorescence microscopy or flow cytometry (Van Denderen et al. (1989); Van Denderen et al. (1992); and Sang et al. (1997)). For example, the ERP-FP1 antibody against the BCR-ABL fusion protein works well in Western blotting procedures but is not successful in microscopic studies on human BCR-ABL-positive leukemias (Van Denderen et al. (1989), and Van Denderen et al. (1992)). Moreover, considering the large variation within individual rearrangements seen in chromosomal translocations and depending on the localization of the breakpoint within the non-aberrant gene (even when the translocations occur within the same two genes) wherein different fusion proteins can be generated, it is deemed likely that within each separate case of fusion proteins, new fusion points arise. Detection of fusion proteins by specific immunologic detection of the fusion-point epitope of the fusion protein has, therefore, never been widely applicable.
An alternative method for the specific detection of fusion proteins involves the application of a so-called catching antibody that recognizes one part of a fusion protein and a labeled detection antibody that recognizes another part of a fusion protein. In such a system, a catching antibody is bound to a solid support layer, such as an ELISA plate or a dipstick. A catching antibody may also be immobilized onto beads that can be analyzed by flow cytometry (P. Berendes (1997)). Following incubation of a catching antibody with a cellular lysate suspected of containing the fusion protein, bound fusion protein is detected by a labeled detection antibody. Although elegant and easy to perform, a catching/detection antibody system cannot be applied practically to detect an intracellular fusion protein without disrupting the cell integrity. Most tumor-specific fusion proteins are localized intracellularly, e.g., nuclear transcription factors, or signaling molecules that reside in the cytoplasm or that shuttle between the cytoplasm and the nucleus. Thus, a catching/detection antibody system does not allow detection of an intracellular fusion protein at the single cell level.
Co-localization of two differentially labeled antibodies against two different parts of a fusion protein could, in theory, prove the presence of a fusion protein in a single cell. However, to full proof co-localization requires analysis by confocal laser scanning microscopy (CLSM). Even then, it is generally not straightforward to evaluate co-localization of two antibodies because the recognized normal proteins that are derived from the normal genes on the unaffected chromosomes can cause a background staining that interferes with the detection of the fusion protein. Further, CLSM has the great disadvantage that it requires a specialized and well-equipped laboratory and trained and highly skilled personnel. Such a time-consuming and highly specialized technique is not desirable for routine diagnostic procedures, e.g., in a clinical setting.
All of the above indicate that there is a specific need for an improved method to detect a fusion protein, which can preferably be used in a clinical laboratory. Particularly challenging is the detection of an intracellular fusion protein at the single cell level.